Abstract In eukaryotes, the biosynthesis of essential Fe-S cluster cofactors is compartmentalized with a mitochondrial iron-sulfur cluster (ISC) machinery and a cytosolic iron-sulfur protein assembly (CIA) machinery. We found that isolated cytosol by itself cannot synthesize Fe-S clusters. Addition of mitochondria to the cytosol, however, allows this process to proceed. Similarly, thiolation of tRNAs is critical for accurate protein synthesis, and isolated cytosol alone cannot thiolate tRNAs but will do so upon addition of mitochondria. We found that the ISC machinery in mitochondria generates two distinct intermediates, Sint and (Fe-S)int. These intermediates are exported to the cytosol by the Atm1 transporter in the mitochondrial inner membrane. Once exported, Sint is utilized for tRNA thiolation and (Fe-S)int is utilized by the CIA machinery for cytosolic Fe-S cluster assembly. Aim 1 is to delineate the pathways for synthesis of Sint vs. (Fe-S)int. The site will be ascertained at which the pathway for Sint formation bifurcates from the ISC pathway for mitochondrial Fe-S cluster synthesis. The site at which (Fe-S)int formation bifurcates will likewise be determined. Mitochondria will be isolated from S. cerevisiae strains lacking components of the ISC machinery and they will be tested in mitochondria-cytosol mixing assays. Aim 2 is to demonstrate a direct role for Atm1 in exporting both Sint and (Fe-S)int. We will determine if the intermediates accumulate in Atm1-depleted mitochondria and if newly imported Atm1 into these mitochondria can restore the export process. Atm1 will be overexpressed to ascertain if this alters competition of Sint and (Fe- S)int for export. The exported and active (Fe-S)int, but not Sint, may contain bound GSH and this will be tested. Aim 3 is to define initial interactor(s) of (Fe-S)int in the cytosol. We will determine whether the glutaredoxins Grx3/4 and/or the Cfd1/Nbp35 scaffold complex interact with purified (Fe-S)int to build their own Fe-S clusters. Aim 4 is to purify and characterize Sint and (Fe-S)int exported from mitochondria. Exported intermediates will be purified by size exclusion chromatography, and active fractions will be characterized by ICP-MS, ESI-MS, and other spectroscopic studies with an aim to identifying the chemical composition of the intermediates. Aim 5 is to define whether mitochondria isolated from mammalian cell lines (CAD or HEK293) also produce and export two distinct intermediates, Sint and (Fe-S)int, and whether they can be swapped for the yeast equivalents in functional assays. Mitochondria lacking ABCB7 (human Atm1) will be tested for their ability/inability to generate Sint and (Fe-S)int intermediates and to export them for cytosolic use. The significance of this work derives from the critical nature of the processes that lie downstream of the exported intermediates. Impaired thiolation of cytosolic tRNAs (downstream of Sint) leads to disruption of protein synthesis. Impaired cytosolic Fe-S cluster assembly (downstream of (Fe-S)int) may create a cellular catastrophe due to genomic instability. Understanding the mechanistic details of mitochondrial production and export of Sint and (Fe-S)int along with detection/identification of these exported species as proposed here is highly significant.